Cell culture media must provide the nutrients necessary to maintain and grow cells in a controlled, artificial and in vitro environment. The particular characteristics of the cell culture media depend to a large extent on the type of cell being cultured and, to a lesser extent on the method of culture.
Mammalian cells have an absolute requirement for iron, which, in vitro, is supplied in the cell culture medium. Bertheussen (Cytotechnology 11:219-231, 1993) has commented that iron cannot be effectively supplied to mammalian cells by adding simple iron salts to the cell culture medium, primarily due to the availability of iron to the cells being reduced by rapid oxidation and precipitation of iron.
Iron in free form, furthermore, has a high oxidative potential, which may result in oxidation of components of a cell culture medium. It has therefore been proven to be of benefit to complex the iron so as to reduce or eliminate this oxidative potential.
In vivo, iron is presented to the mammalian cells by the iron binding protein transferrin. Transferrin works by binding iron and interacting with a transferrin receptor on the cell surface. The transferrin-iron complex is then taken into the cell by endocytosis. Once in the cell, the transferrin-iron complex is broken and the released iron is then complexed to an iron transporting protein (ferritin). The transferrin is recycled. Thorstensen and Romslo, Biochem. J., 271:1-10 (1990) offer an excellent review of this in vivo iron transfer mechanism. The ability of transferrin to mediate transport of iron to cells has been exploited in cell culture by the simple addition of transferrin and an iron salt to the cell culture medium.
However, the transferrin typically used in cell culture media is of animal origin and in recent years there has been increasing regulatory pressure to remove proteins of animal origin from cell culture processes. Clearly the use of proteins of animal origin carries with it the risk of introducing contaminants and adventitious pathogens such as Creutzfeld-Jakob disease (CJD) or Spongiform Encephalopathy (Mad Cow Disease). Alternative iron transporters to transferrin have therefore been sought and applied with varying degrees of success. The type and concentration of any alternative iron transporter has often been found to be dependent on the type of mammalian cell being cultured.
Kovar and Franek (Biotechnology Letters 9:259-264 (1987)) demonstrated that various soluble iron compounds, such as ferric citrate, could be used in place of transferrin in the culture of hybridoma cell lines. Kovar and Franek tested the ability of ferric citrate to support the growth of two hybridoma cell lines over a concentration range of 5 μM (1.25 mg/L) to 5 mM (1225 mg/L). Although lower concentrations of ferric compounds had been proposed in earlier prior art to be suitable for use in culture media for several different cell lines (in particular those of human leukaemic or epithelial origin), Kovar and Franek report that if ferric citrate was to support hybridoma cell growth with equivalence to transferrin, it was required at a concentration of 500 μM (122.5 mg/L). Kovar and Franek found that the medium containing 500 μM ferric citrate was suitable for the culture of other hybridoma cell lines and was also suitable for the culture of several myeloma cell types.
Eto, et al., (Agric. Biol. Chem. 55(3):863-865 (1991)), report similar findings to Kovar and Franek. These workers tested the growth stimulating effect of ferric citrate over a concentration range of 10 mg/L to 600 mg/L on a hybridoma cell line. They report that 300 mg/L was used for further studies. Growth equivalent to that achieved with transferrin was observed when ethanolamine (a lipid precursor) was added at a concentration of 10 μM to the medium containing 300 mg/L ferric citrate.
In a similar study, Toyoda & Inouye, (Agric. Biol. Chem. 55(6):1631-1633 (1991)), tested the growth of three hybridoma cell lines in media containing ferric citrate over a concentration range of 0 to 500 μM. They report that for two of the three hybridoma cell lines tested, 50 μM (12.5 mg/L) ferric citrate was found to be optimal. This result is contrary to the findings of Eto et al., and Kovar and Franek, although a concentration of 500 μM was found optimal for the third cell line.
It is, however, important to note that the work of Kovar & Franek, Toyoda & Inouye, and Eto et al. was all carried out in static culture. WO 94/02592 reports that although 10 mg/L ferric ammonium citrate (FAC) was able to support hybridoma growth in static culture, this was not the case in agitated suspension culture. It is apparent, therefore, that the ability of hybridoma cells to make optimal use of the iron when grown in agitated suspension culture is different from that in static culture.
WO 93/00423 describes a culture medium additive comprising an iron chelate of a soluble iron salt and an alkali metal or alkaline earth metal citrate which is a suitable iron source for serum-free of protein-free culture media. The Examples of this application are concerned predominantly with the growth of mammalian cells such as BHK and CHO cells. Although Example 5 purports to demonstrate the growth of myeloma cells, it is noted that the SP2/0 cells used are in fact non-secreting mouse/mouse hybridoma cells. Culture conditions are specified throughout as being static suspension culture.
Kovar and Franek claim that their medium containing 500 μM ferric citrate was suitable for agitated suspension culture but show no evidence to support this claim. Qi et al., (Cytotechnology 21:95-109 (1996)), however, report that a medium containing 500 μM (122.5 mg/L) ferric citrate, as described by Kovar and Franek, was suitable for the culture of three hybridoma cell lines in agitated suspension cultures. However, Qi et al. found that in order to use a medium containing these high concentrations of ferric citrate (500 μM), it was necessary to wean the cells onto this medium; in the case of one cell line this weaning period was highly protracted. Qi et al. comment that under these conditions the cells were experiencing difficulty adapting and that the medium formulation could be improved by substitution of the ferric citrate in the medium with a more efficient iron presenting compound such as aurin tricarboxlic acid.
In agreement with the prior art cited by Kovar and Franek (1987), several workers have reported that certain types of mammalian cell have been found to be sustainable in culture using lower concentrations of iron compounds.
Ramos et al., WO 92/05246, reports that in the cultivation of epithelial cell lines and in particular Chinese Hamster Ovary (CHO) cell lines, transferrin can be replaced with ferric citrate at 10-100 mg/L (providing approximately 0.6-16 mg/L iron). However, this patent application states clearly that the medium was found not to be suitable for the culture of myeloma cell lines. Keen et al., U.S. Pat. No. 5,633,162, report that ferric citrate, ferrous sulphate and ferric ammonium citrate (FAC) can be used at concentrations of between 0.25 and 5 mg/L (equivalent to 0.04 to 0.8 mg/L iron) to replace transferrin in the culture of CHO cells. WO 98/08934 defines a replacement medium in which all animal proteins, i.e. transferrin and insulin, have been replaced. Transferrin was replaced by ferrous sulphate chelated to a nitrogen-containing chelating compound at concentrations, based on iron, of between 0.28 and 11 mg/L, with 1.1 mg/L being found to be optimal. The nitrogen containing chelating compounds stated as suitable include: ethylenediaminetetraacetic acid (EDTA); ethyleneglycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA); desferoxamine mesylate; diethylenetriaminepentaacetic acid (DTPA) and trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid (CDTA). Of these, EDTA is the most preferred. Ferric citrate was also used in the form of FeCl3-sodium citrate, but this was required at higher concentrations than the ferrous sulphate.7H2O-EDTA chelate.
This application states that the transferrin free medium is suitable for growing mammalian cells, particularly epithelial or fibroblast cells. Exemplification of the growth of CHO and the human embryonic kidney cell line 293 has been provided.
A range of cell types were also tested by Neumannova et al., (In vitro Cell Dev. Biol., 31:625-632 (1995)) for long term growth in media containing iron in the form of ferric citrate at the low concentration of 1.25 mg/L (approximately 0.2 mg/L iron). Of the 19 cell lines tested only 5 were capable of long term growth in this low iron medium. The 5 cell lines were Jurkat, J111 and THP-1 (human leukaemia cell lines), HeLa (a human epithelial cell line) and XC (a rat sarcoma). Although hybridoma and myeloma cell lines were included amongst those tested, none was found to be able to grow in the low iron medium.
As discussed above, lower concentrations of ferric compounds are suitable for use in culture media for certain cell types (particularly those of epithelial and human leukaemic origin). It is, however, generally agreed In the art that, in order to cultivate hybridoma cells in agitated suspension culture using a transferrin free medium, a high concentration, for example in the region of 122.5 mg/L, of an iron compound is required.
The prior art also teaches, however, that high concentrations of iron are not advantageous. Bertheussen, in Cytotechnology 11:219-231(1993), states that high concentrations of iron, such as the 500 μM ferric citrate suggested by Kovar and Franek, should not be used as these high concentrations cause rapid precipitation of iron hydroxide in the medium. Freshly formed iron hydroxide absorbs other metals and various organic molecules efficiently, thus the composition and stability of media containing high iron will be seriously affected.
In view of the difficulties encountered in delivering iron to certain mammalian cells in culture, in particular to hybridoma cells, the concept of chelation of the iron, e.g. to lipophilic compounds was developed.
Iron chelators are typically heterocyclic compounds which attach the metal ion by co-ordinate bonds to at least two non-metal ions in the chelator, and they can be classified using a number of criteria such as their origin (synthetic or biologically produced molecules), their interaction with solvents such as water (hydrophobic vs hydrophilic) or their stochiometric interaction (bidentate of hexadentate).
Lipophilic chelators are compounds which have two distinct properties: (1) the compounds are hydrophobic and often aromatic, thus exhibiting solubility in organic solvents (e.g. alcohol) but limited solubility in water; (2) the compounds also, typically, have a region of negative charge which allows “binding” of iron through electrostatic interactions with positively charged iron ions. In cell culture, it is thought that such compounds will be attracted to the lipid rich membranes of the cells and will, therefore, transport “bound” iron to and possibly through the cell membrane, thus facilitating the supply of iron to the cells (U.S. Pat. No. 5,045,468). The lipophilic chelators are typically added in excess of an accompanying iron salt.
One of the earliest reports of the use of a lipophilic chelator was by Brock and Stevensen (Immunology Letters 15:23-25, (1987)) who used pyridoxal isonicotinoyl hydrazone (PIH) in conduction with ferric nitrilotriacetate. They found that PIH:ferric nitrilotriacetate at a ratio of 2:1 and a concentration of 40 μM (based on PIH) could replace transferrin for the culture of mouse lymphocyte cell lines.
Darfler, (U.S. Pat. No. 5,045,468/in Vitro Cell. Dev. Biol. 26:769-778, 1990) reports a protein free medium suitable for the culture of hybridoma cell lines. The author found that transferrin could be replaced by using the organo iron compound, sodium nitroprusside (SNP) together with EDTA at concentrations of 5.7 and 5.5 mg/L respectively. The author named this medium “ABC medium”.
Bertheussen, (U.S. Pat. No. 5,045,467/Cytotechnology 11:219-231 (1993)), reported that the transferrin in cell culture media could be replaced by using aurin tricarboxlic acid, a lipophilic iron chelator, and 3 μM ferric ions (added in the form of FeCl3). Bertheussen developed this medium using several cell types and found it especially suitable for the culture of fast growing hybridoma cell lines.
WO 94/02592 proposed that tropolone be used to replace the function of transferrin in the presentation of iron to cells in agitated suspension culture. The author comments that tropolone should be added in excess of accompanying iron. The iron may be presented as ferric or ferrous ions using a variety of iron compounds, with FAC the most preferred. A hybridoma cell line was used to elucidate the optimum concentrations of tropolone and FAC as 5 μM and 0.2 mg/L respectively. This medium was also suitable for the growth of NSO myeloma cells. Purely as experimental controls, media lacking transferrin and tropolone but containing FAC were tested with hybridoma and myeloma cells. It was found that FAC alone, between 0.1 and 10 mg/L, was incapable of supporting the growth of hybridoma cells in agitated suspension culture. FAC alone at a concentration of 0.2 mg/L could not support the growth of the NSO myeloma cell lines. No other concentrations of FAC alone were investigated with the myeloma cell lines: presumably the authors assumed that NSO and hybridoma cell lines behave similarly and did not expect other concentrations of FAC to support cell growth.
Keen (Cytotechnology 17:193-202, 1995) reports the development of a protein free medium for the culture of rat myeloma and rat hybridoma cells. This medium, called W38, was based on a 1:1:1 mixture of DMEM, RPMI and the ABC medium developed by Darfler. The medium therefore contained SNP as a lipophilic source of iron and EDTA as a nitrogen containing chelator. SNP and EDTA were, however, at ⅓ of the concentration found in the ABC medium, and Keen found it beneficial to increase the iron concentration by including ferric citrate in the medium. W38 medium was also suitable for the cultivation of the cholesterol auxotrophic myeloma cell line, NS0, providing suitable provision for the cholesterol requirement was made (Keen and Steward, Cytotechnology 17:203-211, 1995).
A recent patent application by Epstein et al., WO 01/16294, comments that in many cases simple iron carriers such as citrate do not provide sufficient iron availability to, or uptake by, cultured cells. The patent also reports that a range of lipophilic iron chelating compounds could be used for a variety of cell types with differing degrees of success. However, results at least as good as transferrin were only obtained with sorbitol chelated to FeCl3 and 2-hydroxypyridine-N-oxide.
The use of lipophilic compounds to chelate and aid presentation of iron in transferrin free culture of hybridoma and myeloma cell types has therefore become state of the art. There are, however, several disadvantages to the inclusion of lipophilic chelators in cell culture medium. The lipophilic chelators are often toxic, for example SNP is classified as highly toxic and has an LD50 in rat of <1 mg/kg. In cell lines used for the industrial production of biotherapeutic products, this has consequences for both manufacturing operators and the final product. Indeed, it may be necessary to develop and validate assays to prove that the final purified biotherapeutic product is clear of any contaminating lipophilic chelator. Additionally, optimisation of the iron concentration of any particular process will be further complicated due to the two-component system of chelator and iron compound.
In summary, the prior art shows that:                1. in the absence of transferrin, hybridoma and myeloma cells will grow in high iron concentrations (122.5 mg/L ferric citrate) (Kovar & Franek).        2. high iron concentrations (e.g. 122.5 mg/L ferric citrate) cause precipitation which damages the culture medium (Bertheussen).        3. in the absence of transferrin or a lipophilic chelator, hybridoma cells will not grow in agitated culture and myeloma cells will not grow at all in low iron concentrations (0.1-10 mg/L and 0.2 mg/L respectively) (WO 94/02592).        4. a lipophilic chelator is required in the medium to enable hybridoma and myeloma cells to grow in agitated culture in low iron concentrations (WO 94/02592).        5. low iron concentrations can be used for growth of certain mammalian cells, but only with the use of a nitrogen-containing chelator such as EDTA (WO 98/08934).        
Within the art of cell culture It is appreciated that certain cell types share similar nutritional attributes. It is notable that both myeloma and hybridoma cell types share significant attributes that are not always exhibited by other cell types, for example glutamine auxotrophy (Bebbington et al. Biotechnology 10:169-175, 1992). The fact that hybridomas and myelomas react in a similar way in many respects is, to a certain extent, unsurprising since hydridoma cell lines are produced by fusion of a myeloma cell with an antibody producing B lymphocyte (Kohler and Milstein, Nature 256:495-497, 1975).
The prior art, as outlined above, has shown that the ability of hybridoma and myeloma cells to use iron present in the medium with a simple iron carrier, such as a citrate, and in the absence of transferrin or a lipophilic or nitrogen-containing chelator is different from the corresponding ability of, for example, CHO cells. The overall teaching of the prior art is that hybridoma and myeloma cells are the same in their ability to use iron in a transferrin-free culture medium.
It is apparent from the literature, as discussed above, that time and effort have been dedicated to the development of transferrin free media for the growth of hybridoma cell types, particularly in static culture. On the art-implied assumption that myeloma cells will demonstrate the same requirements as hybridoma cells in this respect, an equivalent effort has not been made with myeloma cells.
Over the years, it is apparent that the art of hybridoma and myeloma cell culture has moved away from the use of iron in the form of a soluble iron compound as a replacement for transferrin, and instead has determined that to reap the benefits of low iron concentrations, a lipophilic or nitrogen-containing chelator must be included in a culture medium. Of course the use of such chelators in media used to grow cells for the product they have been engineered to produce, also necessitates that the product is further treated to ensure there is no contamination with that chelator. The inclusion of such a chelator in the culture medium thus not only extends the time taken to demonstrate that the product is pure, but also increases the expense of production by necessitating development of specific assays to prove that a “toxic” iron chelator does not contaminate the pure product.
It is apparent, therefore, that a need still exists to provide iron in a simple form to certain cells in culture at concentrations which will provide sufficient iron to enable continuing cell growth but which will not result in the precipitative damage discussed above. A further advantage would be to have a medium free of transferrin and lipophilic or nitrogen-containing chelators. The aim would be to achieve equivalent cell culture in a transferrin and lipophilic or nitrogen-containing chelator-free medium as would be obtained in a medium containing transferrin.